Prof. Enrico Gratton - Lecture 6
Fluorescence Microscopy
Instrumentation
Light Sources:
One-photon and Multi-photon Excitation
Applications in Cells
Lifetime Imaging
Figures acknowledgements: E.D. Salmon and K. Jacobson
Confocal microscopy images
In the compound microscope the Finite Corrected
Objective Forms a Real Image At the Ocular Front Focal
Plane: The Primary or Intermediate Image Plane (IIP)
PPL
S1
O
2
F
1
F'
3
I
s2
fob
fob
foc
OTL
IIP
Conventional Optics
Objective with finite Focal Length
(Optical Tube Length, OTL, Typically 160 mm)
Mob = OTL/fob
Total Magnification = Mob x Moc = OTL/fob x 250mm/foc
Why is the eyepiece necessary?
E.D. Salmon
Resolution Limitations of the Human Eye
Limits to Accommodation
O
O'
O"
α
β
I
γ
I'
Unresolved
I"
Resolved
250 mm
Conventional
Viewing
Distance
³ 2.0 µm
Resolution Test
COARSE
A
B
FINE
A word about
infinity corrected
optics and its
advantages.
Modern microscope component identification
Prisms Used to
Re-Direct Light
In Imaging Path
While Mirrors
Are Used in
Illumination
Path
E.D.Salmon
MICROSCOPE COMPONENTS
Camera
Identify
Major
Components
And
Their
Locations
And Functions
Within
Modern
Research Light
Microscope
(See Salmon
And Canman,
2000, Current
Protocols in Cell
Biology, 4.1)
Binocular
Camera Adapter
Eyepiece
Epi-Condenser
Diaphragm
Beam Switch
Magnification
Changer
Epi-Field
Diaphragm
& Centering
Filters
Epi-Lamp Housing
Shutter
Mirror:
Focus and
Centering
Filter Cube
Changer
Slot for Analyzer
Body Tube
Focus, Centering
Slot for DIC Prism
Objective Nosepiece
Objective
Stage
Trans-Lamp Housing
Condenser:
Diaphragm&Turret
Centering
Focus
Mirror:
Focus and
Centering
Slot for Polarizer
Field Diaphragm
Upright Microscope
Stand
Coarse/Fine
Specimen Focus
Filters
and Diffuser
Lamp: Focus, Centering
Key component: the objective
Achromats: corrected for chromatic aberration for red, blue
Fluorites: chromatically corrected for red, blue; spherically
corrected for 2 colors
Apochromats: chromatically corrected for red, green & blue;
spherically corrected for 2 colors
Plan-: further corrected to provide flat field
The 3 Classes of Objectives
Chromatic and Mono-Chromatic Corrections
E.D. Salmon
What is numerical aperture (NA)?
• Image Intensity: I ~ NAobj2/Mtot2
• Image Lateral Resolution for
Corrected Objective:
-Fluorescence: r = 0.61λ/NAobj
-Trans-Illumination: r = λ/(NAobj +
NAcond)
I
Airy Disk Formation by Finite
Objective Aperture:
The radius of the Airy Disk at
the first minimum, r’, occurs
because of destructive
interference; the diffraction
angle, α, is given by:
sin(α) = 1.22λ/D, where D =
diameter of objective back
aperture
r'
x
α
j
i
E.D. Salmon
O
Lateral Resolution in Fluorescence
Depends on Resolving Overlapping
“Airy Disks”
Rayleigh Criteria: Overlap by r’,
then dip in middle is 26% below
Peak intensity
(2πx/λ)NAobj
E.D.Salmon
E.D. Salmon
Resolution is better at shorter wavelengths,
higher objective NA or higher condenser NA
High NA and/or shorter λ
Low NA and/or longer λ
Rayleigh Criterion for the resolution of two adjacent spots:
Plim = 0.61 λo / NAobj
Examples: (λ
λo = 550 nm)
Mag
high dry 10x
40x
oil
100x
63x
f(mm)
16
4
1.6
2.5
n
1.00
1.00
1.52
1.52
a
NA
Plim (µm) (NAcond=NAobj)
15
40
61
67.5
0.25
0.65
1.33
1.40
1.10
0.42
0.204
0.196
Why oil immersion lenses have greater resolution
D= 0.61 λ cos α / n(NA)2
Low power, NA~ 0.25
Hi, dry,
NA~0.5
Oil immersion, NA~ 1.3
D~ 8 µm
D~ 2 µm
D~0.4 µm
Contrast : All the resolution in the world won’t do
you any good, if there is no contrast
to visualize the specimen.
CONTRAST = (Isp - Ibg )/Ibg
HIGH
LOW
E.D.Salmon
1
2
3
4
5
6
7
8
9
10
Fluorescence
Index of refraction
Brightfield
Phase contrast
Brightfield
Normalized interference
Darkfield
Darkfield
Basic design of the epi fluorescence microscope
Objectives
High transmittance
Fluorite lenses: λ > 350 nm [ok for FURA]
Quartz lenses: λ < 350 nm
Employ simple, non plan lenses to minimize
internal elements.
Negligible auto-fluorescence or solarization [color
change upon prolonged illumination]
Maximizing image brightness (B)
excitation efficiency ~ (NA)2
=> B ~ (NA)4
collection efficiency ~ (NA)2
(NA)4
1
also B ~
=>
M2
at high NA,
B~
, for NA ≤ 1.0
M2
Filters
Interference filter definitions
Filter cube
designs
employing
longpass emitter
filters
Filter cube
designs
employing
bandpass emitter
filters
Multi-Wavelength Immunofluorescence
Microscopy
PIXELS
The building blocks of CCDs
Back thinned CCDs receive light from this side
Primary Features of CCD
•
•
•
Spatial resolution of the CCD array
– Number of Pixels in X and Y
– Center to Center Distance of Pixels in microns
Full Well Capacity
– Related to Physical size and electronic design
– Determines Maximum Signal level possible
Quantum Efficiency/Spectral Range
– Determines the usefulness of the camera
– Major influence on exposure time
• Camera Noise
– The limiting feature in low light applications
– Influenced by Readout Speed / Readout Noise
– Influenced by Dark Current / Time
• CCD Chip Design
– Influences Total Frame Rate
• Exposure time plus Readout time
– Total Photon Efficiency
• Quantum Efficiency and Exposure Cycle
B. Moomaw, Hamamatsu Corp.,
Types of CCD Detectors
• CCD Cameras - 3 Primary Designs
B. Moomaw, Hamamatsu Corp.
Improvements in Interline CCDs
• Effective Q.E. was greatly increased by Microlens
technology.
Single microlens added
Input
light
Old IT CCD
B. Moomaw, Hamamatsu Corp.
Open window
Microlens
Latest Improvement to Interline
CCDs
• Latest double micro lens structure improved the CCD
open ratio up to 80% and Q.E. to over 70%!
Input light
Double lens
structure added
B. Moomaw, Hamamatsu Corp.
Noise as a function of incident
camera illumination
(Camera Noise =10 electron, QE =0.4)
1000
Total Noise
NSignal » Ncamera
Shot Noise
Camera Noise
100
NCamera » NSignal
10
S/N = S/NCamera
S/N = S/NSignal =
S
1
1
10
100
1,000
Inp ut Light Le ve l (pho ton)
10,000
100,000
COMMON SOURCES OF AUTOFLUORESCENCE
Autofluorescent Source
Typical Emission Wavelength (nm)
Typical Excitation Wavelength (nm)
Flavins
520 to 560
380 to 490
NADH and NADPH
440 to 470
360 to 390
Lipofuscins
430 to 670
360 to 490
Advanced glycation
end-products (AGEs)
385 to 450
320 to 370
Elastin and collagen
470 to 520
440 to 480
530
488
685 (740)
488
Lignin
Chlorophyll
From Biophotonics International
Photobleaching
• Photochemical lifetime: fluorescein will
undergo 30-40,000 emissions before bleaching.
(QYbleaching ~ 3*10-5)
• At low excitation intensities, photobleaching
occurs but at lower rate.
• Bleaching is often photodynamic--involves light
and oxygen.
Parameters for Maximizing
Sensitivity
• Use High Objective NA and Lowest Magnification:
Ifl ~ IilNAobj4/Mtot2
-Buy the newest objective: select for best
efficiency
• Close Field Diaphragm down as far as possible
• Use high efficiency filters
• Use as few optical components as possible
• Match magnification to camera resolution:
MMax = 3*Pixel Size of Detector/Optical
Resolution
E.g.: 3*7 µm/[0.6 *520nm/1.4] = 91X
• Reduce Photobleaching
• Use High Quantum Efficiency Detector in Camera
Adapted from E.D.Salmon
Live Cell Considerations
• Minimize photobleaching and photodamage
(shutters)
• Use heat reflection filters for live cell imaging
• Image quality: Maximize sensitivity and signal to
noise (high transmission efficiency optics and high
quantum efficiency detector)
• Phase Contrast is Convenient to Use with EpiFluorescence
– Use shutters to switch between fluorescence and
phase
– Phase ring absorbs ~ 15% of emission and slightly
reduces resolution by enlarging the PSF
Adapted from E.D. Salmon
Defining Our Observation Volume:
One- & Two-Photon Excitation.
2 - Photon
1 - Photon
Defined by the pinhole size,
wavelength, magnification
and numerical aperture of
the objective
Approximately 1 um3
Defined by the wavelength
and numerical aperture of
the objective
Advantages of two-photon excitation
Brad Amos
MRC, Cambridge, UK
3-D sectioning effect
Absence of photo bleaching in out of focus regions
Large separation of excitation and emission
No Raman from the solvent
Deep penetration in tissues
Single wavelength of excitation for many dyes
High polarization
Why confocal detection?
Molecules are small, why to observe a large volume?
• Enhance signal to background ratio
• Define a well-defined and reproducible volume
Methods to produce a confocal or small volume
(limited by the wavelength of light to about 0.1 fL)
• Confocal pinhole
• Multiphoton effects
2-photon excitation (TPE)
Second-harmonic generation (SGH)
Stimulated emission
Four-way mixing (CARS)
(not limited by light, not applicable to cells)
• Nanofabrication
• Local field enhancement
• Near-field effects
How does one create an observation volume and collect the data?
Two-Photon, Scanning, FCS Microscope
Sample
Mirror
Scanner
Titanium Sapphire Laser
Mode-Locked 150 fs pulses
Microscope
Argon Ion Laser
Em1
Dichroic BS
Detector
Detector
Em2
Computer
Laser technology needed for two-photon excitation
Ti:Sapphire lasers have pulse duration of about 100 fs
Average power is about 1 W at 80 MHz repetition rate
About 12.5 nJ per pulse (about 125 kW peak-power)
Two-photon cross sections are typically about
δ=10-50 cm4 sec photon-1 molecule-1
Enough power to saturate absorption in a diffraction limited spot
2
d pπ A 2
)
na ≈ (
τ fhcλ
na
p
τ
f
A
λ
d
Photon pairs absorbed per laser pulse
Average power
pulse duration
laser repetition frequency
Numerical aperture
Laser wavelength
cross-section
Intensity
exc
Laser 2-photon
em
Raman
400
600
Wavelength (nm)
800
Fluorescein
Rhodamine B(MeOH)
Laurdan(MeOH)
Rhodamine 110(MeOH)
Rhodamine 123(MeOH)
100
80
4
η 2σ 2 (10 cm s/photon)
120
-50
60
40
20
0
720
740
760
780
800
wavelength nm
820
840
860
MEQ (H2O)
ANS (MeOH)
25 Dansyl Chloride (MeOH)
POPOP (MeOH)
General References
• Salmon, E. D. and J. C. Canman. 1998. Proper
Alignment and Adjustment of the Light Microscope.
Current Protocols in Cell Biology 4.1.1-4.1.26, John
Wiley and Sons, N.Y.
• Murphy, D. 2001. Fundamentals of Light Microscopy
and Electronic Imaging. Wiley-Liss, N.Y.
• Keller, H.E. 1995. Objective lenses for confocal
microscopy. In “Handbook of biological confocal
microsocpy”, J.B.Pawley ed. , Plenum Press, N.Y.
On line resource:
Molecular Expressions, a Microscope
Primer at:
http://www.microscopy.fsu.edu/primer/
index.html